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Abstract

Background

To study the expression of MK-1 and RegⅣ and to detect their pathological significances
in benign and malignant lesions of gallbladder.

Keywords:

Introduction

MK-1, also known as Ep-CAM, is a type-I transmembrane protein with cell adhesion activity
expressed on normal epithelial cells of various tissues including stomach, colon,
pancreas, lung, breast and ovary. MK-1 has been suggested to be involved in the differentiation
and growth of epithelial cells under normal physiological conditions through its homotypic
cell-cell adhesion activity [1-5]. Because it is over-expressed on most carcinomas, MK-1 has been used as a target
for diagnosis and therapy of cancer [1-5]. The regenerating gene (Reg) family, a group of small secretory proteins, is involved
in cell proliferation or differentiation in digestive organs, is upregulated in several
gastrointestinal cancers, and functions as trophic or anti-apoptotic factors [6-11]. RegIV, a member of the regenerating gene family, is involved in digestive tract
malignancies, including the stomach, colorectum, and pancreas, as well as in benign
diseases such as ulcerative colitis [6-11]. RegIV overexpression in tumor cells has been associated with cell growth, survival,
adhesion, and resistance to apoptosis [6-11]. Although the expression of MK-1 has been reported in carcinomas of various origins,
only one study has described its expression in gallbladder adenocarcinoma. No study
on the expression of RegIV in gallbladder adenocarcinoma has been published. In the
present study, we studied the expression of MK-1 and RegⅣ in various benign and malignant
lesions of gallbladder, and evaluated their prognostic usefulness.

Materials and methods

Patients and clinical information

A total of 204 specimens, including 108 adenocarcinomas, 46 peritumoral tissues, 35
chronic cholecystitis tissues, and 15 gallbladder polyp, were studied ethically with
pre-approval from Ethics Committee of Human Study of Central South University. All
of these samples were collected between 1996 and 2006. Among the 108 adenocarcinoma,
31 cases are male (28.7%) and 77 cases are female (71.3%) with an average age of 52.6
± 11.2 years. Diagnoses of adenocarcinomas were based on morphological criteria, immunohistochemical
staining, and the clinical findings. The histopathologic subtypes of the 108 adenocarcinomas
include 36 well-differentiated adenocarcinomas (33.3%), 31 moderately-differentiated
adenocarcinomas (28.7%), 30 poorly-differentiated adenocarcinomas (27.8%), and 11
mucinous adenocarcinomas (10.2%). The invasion was evaluated according to the standard
criteria for T-stages [12]. Among the 108 adenocarcinomas, 14 cases are T1, 35 cases are T2, 37 cases are T3,
and 22 causes are T4 stage. Invasion of gallbladder surrounding tissues and organs
was found in 59 patients (54.6%) with adenocarcinoma while 59 patients had regional
lymph node metastasis (54.6%). 58 cases had gallstones (53.7%). Surgery included radical
resection for 34 adenocarcinoma (31.5%), palliative surgery for 48 adenocarcinomas
(44.4%) and no resection operation for 26 cases (24.1%) (The specimens were obtained
with surgical biopsy in these patients). Survival information of 67 cases among the
108 adenocarcinomas was obtained through letters and phone calls. Among them, 20 cases
survived over 1 year and 47 cases survived less than 1 year. The chronic cholecystitis,
gallbladder polyp and peritumoral tissues were diagnosed according to the published
standard criteria [13]. The 46 peritumoral tissues (distance from cancer ≥ 3 mm) were obtained from the
108 patients with adenocarcinomas. Among them, 10 tissues are normal, 10 tissues are
mild dysplasia, 12 tissues are moderate dysplasia, and 14 tissues are severe dysplasia.
Among the 35 cases with chronic cholecystitis, 15 cases are males (42.9%) and 20 are
females (57.1%). According to the criteria for dysplasia described by Dowling and
Kelly [14], the 35 cases of chronic cholecystitis (15 have chronic cholecystitis only and 20
have chronic cholecystitis accompanied by gallbladder stone) were classified into
normal and mild, moderate, and severe dysplasia: 11 cases without cellular atypia
as normal mucosa, 12 cases with mild cellular atypia as mild dysplasia, 7 cases with
moderate cellular atypia as moderate dysplasia, and 5 cases with severe cellular atypia
as severe dysplasia. Among the 15 polyps, 10 were confirmed to have epithelial normal
to mild dysplasia by pathological examination and 5 have moderate to severe dysplasia.

Pathological evaluation

Stained slides were evaluated in a blinded manner in all cases for membrane and cytoplasm
staining. MK-1 and RegIV expression was defined as the presence of specific staining
on the surface membranes and cytoplasm of tumor cells. MK-1 and RegIV expression was
considered as positive when over 25% of tumor cells exhibited a positive reaction
[13,15].

Statistical analysis

Data was analyzed using the statistical package for the Social Sciences Version 13.0
(SPSS 13.0). The inter-relationship of MK-1 or RegIV expression with histology or
clinical factors was analyzed using χ2 or Fisher's exact test. Kaplan-Meier and log-rank test were used for univariate survival
analysis. Cox proportional hazards model was used for multivariate analysis and to
determine the 95% confidence interval. Results were considered significant if a two-sided
P value of less than 0.05 was obtained.

Results

MK-1 and RegIV expression in benign and malignant lesions of gallbladder

The expression of MK-1 and RegIV was primarily located in the membrane and cytoplasm
(Figure 1). The positive expression of MK-1 and RegIV in gallbladder adenocarcinoma samples
was significantly higher than in peritumoral tissues, polyp or chronic cholecystitis
(P < 0.01) (Table 1). Among the 108 gallbladder adenocarcinoma samples, 67 of them were MK-1 positive
(62.0%) and 58 were RegIV positive (53.7%). The positively expressed peritumoral tissue,
polyp and chronic cholecystitis showed atypical hyperplasia of epitheli.

Table 1. Relationship between ANXA1 and ANXA2 expression and the lifetime of gall bladder adenocarcinoma

MK1 and RegIV expression is associated with the clinicopathological characters of
adenocarcinoma

In adenocarcinomas that were highly differentiated, with tumor diameter < 2 cm, with
no lymph node metastasis and no peritissue invasion, MK-1 positive expression rate
was significantly higher than those that were moderately- or low-differentiated, with
tumor maximum diameter ≥ 2 cm, with lymph node metastasis and peritissue invasion
(P < 0.05). The expression of RegIV showed opposite pattern (P < 0.05). The expression of MK-1 and RegIV showed no obvious relation with other clinicopathological
characters, such as age, sex or cholecystolithiasis (P > 0.05) (Table 2).

Table 2. Relationship between MK-1 and RegIV expression and the clinical pathology characters
of adenocarcinoma

Relationship between MK-1 and RegIV expression and the lifetime of patients with gall
bladder adenocarcinoma

According to survey by mail or phone (2 years), we got 67 profiles from the 108 gallbladder
adenocarcinoma, 20 of them survived more than or equal to 1 year after surgery, 47
of them had less than 1 year life cycle, and mean life cycle was 9.6 ± 5.2 months.
Among the 67 cases, the positive expression of MK-1 and RegIV was 38 (56.7%) and 34
(50.7%). After Kaplan-Meier survival analysis, we found that the mean survival time
after surgery was closely related to pathological types (P = 0.031), maximum diameter of tumor (P = 0.003), lymphatic metastasis status and peritissue invasion (P = 0.002). The mean survival time after surgery was significantly higher in the MK-1
positive cases than the negative ones (P = 0.009), while the mean survival time after surgery was significantly lower in the
RegIV positive cases than the negative ones (P = 0.003) (Figure 2). Table 3 summarizes the results of multivariate survival analysis using the Cox proportional
hazards model. Multivariate analysis revealed that maximum diameter of tumor ≥ 2 cm,
lymphatic metastasis and peritissue invasion, MK-1 negative expression and RegIV positive
expression were negatively correlated with mean survival time after surgery, positively
correlated with mortality, and are independent prognostic markers.

Figure 2.MK1 and RegIV expression and survival in patients with adenocarcinoma of gallbladder. A, Kaplan-Meier plots of overall survival in patients with gallbladder adenocarcinoma
and with MK1 positive and negative expression. B, Kaplan-Meier plots of overall survival
in patients with gallbladder adenocarcinoma and with RegIV positive and negative expression.

Discussion

Our study shows the prognostic significance of MK-1 and RegIV expression as a tumor
biological marker in patients with gallbladder adenocarcinoma. We found that positive
expression rate of MK-1 and RegIV were higher in gallbladder adenocarcinoma than those
in peritumoral tissues, polyp and chronic cholecystitis. Statisitical analysis revealed
that MK-1 negative expression and RegIV positive expression is negatively correlated
with mean survival time after surgery, positively correlated with mortality, and are
independent prognostic markers.

The expression of MK-1 has been previously reported in several carcinomas. In gastric
carcinomas, MK-1 is more frequently expressed in cardiac tumors (with 50% expression
rate), in large (> 3 cm) tumors, and in specimens from patients with more than five
metastatic lymph nodes [2]. In urinary bladder carcinoma, 56.8% were positive for MK-1 protein expression and
significant correlations were observed between MK-1 expression and tumor grade, schistosoma,
DNA ploidy and tumor recurrence [3]. In Gallbladder carcinoma, expression of MK-1 was found in 50 (79%) of 63 tumor samples.
Multivariate analysis showed that MK-1 expression was an independent prognostic marker,
and Kaplan-Meier curves showed that MK-1 expression was significantly related to increased
overall survival, suggesting that MK-1 expression is a prognostic marker in gallbladder
carcinoma [4]. In our study the positive expression rate of MK-1 was slightly lower than what was
previously reported (62% vs 79%), however, our statistical analysis revealed similar
prognostic significance of MK-1 expression.

The expression of RegIV has also been reported to be increased in some carcinomas
including prostate, pancreatic and gastric cancer [6-11]. The expression of RegIV has been found to be related to the carcinogenesis, clinical
biological behaviors, and prognosis of the carcinomas studied. Most of the high RegIV
cases have poorly differentiated, high clinical stage, prone to metastasis and strong
invasion ability, which are all considered being bad indicators of cancer prognosis
[6-11,16]. These results all support that RegIV may involve in cancer formation and affect
its development and prognosis. To the best of our knowledge our study is the first
of RegIV expression in gallbladder adenocarcinoma. We showed that the positive expression
of RegIV in gallbladder adenocarcinoma samples was significantly higher than in benign
gallbladder lesions. In adenocarcinomas, MK-1 positive expression rate was significantly
higher in samples that were highly differentiated, with tumor diameter < 2 cm, with
no lymph node metastasis and no peritissue invasion. Moreover, RegIV positive expression
was negatively correlated with mean survival time after surgery, positively correlated
with mortality, and are independent prognostic markers. These findings indicate that
RegIV is a promising tumor marker in gallbladder adenocarcinoma.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

LY and ZY designed the study, and performed the experiment and statistical analysis
and drafted the manuscript. SL participated in the immunostaining. JL participated
in the statistical analysis. All authors read and approved the final manuscript.